Y-box结合蛋白1对肝祖细胞介导的肝组织再生和肝纤维化形成的调节机制研究

Hepatic progenitor cells（HPC） participant in liver regeneration during chronic liver injury, and its activation and proliferation is also closely related with liver fibrogenesis.Y-box binding protein 1 (YB-1) is a secreted multifunctional protein.Previously studies have shown that YB-1 was involved in the fibrogenesis of many tissues and organs with highly cell and tissue specificity.Our project intend to study the impact of down and up-regulated YB-1 by siRNA silencing and plasmid over-expression technologies on the proliferation,differentiation of HPC，and the activation, proliferation, migration, extracellular matrix synthesis of hepatic stellate cells（HSC）. Acute and chronic liver injury models in YB-1 transgenic knockout mice are constructed， and the effect of YB-1 on liver regeneration and fibrogenesis is clarified.Finally,we will investigat YB-1 expression in different causes of chronic liver disease, and clarify the association of YB-1 between HPC and HSC in different conditions.The results will further elucidate the mechanism of YB-1 in the process of liver regeneration and fibrogenesis in chronic liver injury, thus help to explore the molecular mechanism of liver fibrosis from a new perspective and provide new theoretical and experimental basis for clinical treatment of liver fibrosis.

肝祖细胞（HPC）作为慢性肝损伤时肝脏修复重要参与者，其活化增殖反应与纤维化发生和发展关系密切。Y-box结合蛋白1（YB-1）是一种可分泌多功能蛋白，前期研究显示其参与了多种组织、器官纤维化进程，且其发挥作用具有明显的细胞和组织特异性。本课题拟从细胞水平用siRNA干扰和质粒过表达方式下调和上调HPC与肝星状细胞（HSC）中YB-1，探索其对HPC增殖和分化以及HSC多种生物学功能的影响。并在YB-1转基因敲除鼠中构建急、慢性肝脏损伤修复模型，从体内角度阐明YB-1对肝脏损伤修复和纤维化形成影响。最后，观察不同病因慢性肝病中YB-1表达量和分布变化情况，阐明不同病因条件下YB-1与HPC以及HSC之间的关联。研究结果将进一步阐明YB-1在慢性肝脏损伤过程中肝脏修复和纤维化形成作用机制，从而有助于从YB-1这一新视角探索肝纤维化分子机制，为临床治疗肝纤维化提供新的理论和实验依据。

肝祖细胞（HPC）作为慢性肝损伤时肝脏修复重要参与者，其活化增殖反应与纤维化发生和发展关系密切。Y-box结合蛋白1（YB-1）是一种可分泌多功能蛋白，研究表明其参与多种组织、器官纤维化进程，且其发挥作用具有明显的细胞和组织特异性。. 本课题采用免疫荧光和免疫组化技术检测发现，正常人肝脏不表达YB-1，慢性乙型肝炎患者肝脏高表达YB-1，其中又以胆管细胞和肝祖细胞升高最为明显，并与肝纤维化程度呈正相关；ChIP-sequence 和RNA-sequence均提示YB-1能调控Hedgehog信号通路；ChIP-sequence和ChIP-PCR表明YB-1能与Gli2启动子区结合；荧光素酶报告基因实验表明YB-1能调控Gli2的转录；沉默YB-1能抑制Gli2的表达，通过阻断Hedgehog信号通路而抑制HPC增殖；ChIP-sequence 和RNA-sequence提示YB-1能调控细胞粘附和细胞外基质相互作用；RT-PCR、Western blot和免疫荧光均表明肝祖细胞能分泌细胞外基质；ChIP-sequence 和ChIP-PCR表明YB-1能与PDGFR-β启动子区结合并负调控其转录；荧光素酶报告基因实验表明YB-1能负调控PDGFR-β的转录；沉默YB-1能促进HPC表达细胞外基质，并且PDGFR-β/ERK/p90RSK信号通路参与该过程。综上所述，YB-1能通过Hedgehog信号通路调控肝祖细胞增殖，通过PDGFR-β/ERK/ p90RSK信号通路负调控肝祖细胞分泌细胞外基质。. 此外，该研究还表明正常人和正常小鼠肝组织不表达YB-1，低表达Wnt3;慢性乙型病毒性肝炎相关肝硬化患者及小鼠肝损伤模型的肝组织高表达YB-1 和Wnt3，且在胆管反应区最为显著，并与肝纤维化程度呈正相关。体外实验表明HPC 可定向分化为肝细胞。ChIP-sequence 与ChIP-PCR 证实YB-1 在Wnt3 启动子区存在结合位点，生物信息学分析发现该结合位点在Wnt3 基因转录起始点上游5’UTR39366bp 处。荧光素酶酶报告基因显示YB-1 能负性调节Wnt3 启动子的表达活性，进而调控Wnt/β-catenin 信号通路进而调节HPC 向肝细胞分化。